Inhibition of Monoamine Oxidases by Pyridazinobenzylpiperidine Derivatives

Monoamine oxidase inhibitors (MAOIs) have been crucial in the search for anti-neurodegenerative medications and continued to be a vital source of molecular and mechanistic diversity. Therefore, the search for selective MAOIs is one of the main areas of current drug development. To increase the effectiveness and safety of treating Parkinson’s disease, new scaffolds for reversible MAO-B inhibitors are being developed. A total of 24 pyridazinobenzylpiperidine derivatives were synthesized and evaluated for MAO. Most of the compounds showed a higher inhibition of MAO-B than of MAO-A. Compound S5 most potently inhibited MAO-B with an IC50 value of 0.203 μM, followed by S16 (IC50 = 0.979 μM). In contrast, all compounds showed weak MAO-A inhibition. Among them, S15 most potently inhibited MAO-A with an IC50 value of 3.691 μM, followed by S5 (IC50 = 3.857 μM). Compound S5 had the highest selectivity index (SI) value of 19.04 for MAO-B compared with MAO-A. Compound S5 (3-Cl) showed greater MAO-B inhibition than the other derivatives with substituents of -Cl > -OCH3 > -F > -CN > -CH3 > -Br at the 3-position. However, the 2- and 4-position showed low MAO-B inhibition, except S16 (2-CN). In addition, compounds containing two or more substituents exhibited low MAO-B inhibition. In the kinetic study, the Ki values of S5 and S16 for MAO-B were 0.155 ± 0.050 and 0.721 ± 0.074 μM, respectively, with competitive reversible-type inhibition. Additionally, in the PAMPA, both lead compounds demonstrated blood–brain barrier penetration. Furthermore, stability was demonstrated by the 2V5Z-S5 complex by pi–pi stacking with Tyr398 and Tyr326. These results suggest that S5 and S16 are potent, reversible, selective MAO-B inhibitors that can be used as potential agents for the treatment of neurological disorders.


Introduction
Parkinson's disease (PD) is a neurodegenerative disease that is particularly common among the elderly and is characterized by the death of dopaminergic neurons in the substantia nigra pars compacta [1,2].The typical motor features of PD (bradykinesia, stiffness, and tremor) are caused by a subsequent dopamine shortage; however, non-dopamine neurodegeneration results in additional non-motor symptoms that manifest at different times [3].The pathogenesis of the disease is characterized by elevated acetylcholine and low dopamine levels; therefore, treatment strategies aimed at increasing dopamine levels are recommended.One such strategy is the inhibition of monoamine oxidases (MAOs), which are responsible for the metabolism of endogenous amines (Figure 1) [4][5][6][7].
Molecules 2024, 29, 3097 2 of 19 low dopamine levels; therefore, treatment strategies aimed at increasing dopamine levels are recommended.One such strategy is the inhibition of monoamine oxidases (MAOs), which are responsible for the metabolism of endogenous amines (Figure 1) [4-7].MAO inhibitors (MAOIs) have been crucial in the search for anti-neurodegenerative medications and continue to be a vital source of molecular and mechanistic diversity [7].Therefore, the search for selective MAOIs is one of the main areas of current drug development.To increase the effectiveness and safety of treating PD, new scaffolds for reversible human MAO-B (MAO-B) inhibitors are being developed [8][9][10][11].
Selegiline and rasagiline are MAO-B inhibitors used to treat levodopa-induced motor fluctuations, advanced PD, and the loss of OFF time in patients.Safinamide, a third MAO-B inhibitor, is used as an adjuvant therapy or as a dopamine agonist in conjunction with levodopa, and it combines additional non-dopaminergic qualities that may be beneficial for patients with PD (Figure 2) [12][13][14].The pharmacological properties of pyridazinone analogs include anti-inflammatory, anticancer, antibacterial, anticonvulsant, analgesic, antioxidant, antihypertensive, antisecretory, and antiulcer effects [13][14][15][16][17][18][19][20][21].They include lipoxygenase inhibitors, cholinesterase MAO inhibitors (MAOIs) have been crucial in the search for anti-neurodegenerative medications and continue to be a vital source of molecular and mechanistic diversity [7].Therefore, the search for selective MAOIs is one of the main areas of current drug development.To increase the effectiveness and safety of treating PD, new scaffolds for reversible human MAO-B (MAO-B) inhibitors are being developed [8][9][10][11].
Selegiline and rasagiline are MAO-B inhibitors used to treat levodopa-induced motor fluctuations, advanced PD, and the loss of OFF time in patients.Safinamide, a third MAO-B inhibitor, is used as an adjuvant therapy or as a dopamine agonist in conjunction with levodopa, and it combines additional non-dopaminergic qualities that may be beneficial for patients with PD (Figure 2) [12][13][14].
Molecules 2024, 29, 3097 2 of 19 low dopamine levels; therefore, treatment strategies aimed at increasing dopamine levels are recommended.One such strategy is the inhibition of monoamine oxidases (MAOs), which are responsible for the metabolism of endogenous amines (Figure 1) [4-7].MAO inhibitors (MAOIs) have been crucial in the search for anti-neurodegenerative medications and continue to be a vital source of molecular and mechanistic diversity [7].Therefore, the search for selective MAOIs is one of the main areas of current drug development.To increase the effectiveness and safety of treating PD, new scaffolds for reversible human MAO-B (MAO-B) inhibitors are being developed [8][9][10][11].

Chemistry
This study synthesized and investigated the in vitro enzyme inhibitor activities of 23 compounds that act as selective MAO-B inhibitors, which were obtained from 6substituted-3(2H)-pyridazinone-2-acetyl-2-(substituted benzalhydrazone) [10,11].Besides unsubstituted benzaldehyde, benzaldehyde derivatives with disubstituted methylamine, methyl, bromo, fluoro, methyl, and methoxy substituents at various positions were used in synthesizing benzhydrazone derivatives, and the corresponding contributions of these groups to the activity were investigated.The structures of the obtained compounds were confirmed using spectrum data from 1H-NMR, 13C-NMR, and TOF-MS.Their melting temperatures were determined, and the findings are shown in the Supplementary File and Table 1.methyl, bromo, fluoro, methyl, and methoxy substituents at various positions were used in synthesizing benzhydrazone derivatives, and the corresponding contributions of these groups to the activity were investigated.The structures of the obtained compounds were confirmed using spectrum data from 1H-NMR, 13C-NMR, and TOF-MS.Their melting temperatures were determined, and the findings are shown in the Supplementary File and Table 1.Twenty-four pyridazinobenzylpiperidine derivatives against MAO-A and MAO-B were evaluated.These pyridazinobenzylpiperidine derivatives were synthesized with two large structures: one residue was attached to the phenyl ring, and two or more residues were attached to the phenyl ring (Figure 4).Seventeen of the twenty-four compounds showed higher MAO-B inhibition than MAO-A except seven compounds (Table 2).Compound S5 showed potent MAO-B inhibition with an IC50 value of 0.203 µM, followed by S16 (IC50 = 0.979 µM).In contrast, compound S15 showed the highest MAO-A inhibition with an IC50 value of 3.691 µM, followed by S5 (IC50 = 3.857 µM) ( Twenty-four pyridazinobenzylpiperidine derivatives against MAO-A and MAO-B were evaluated.These pyridazinobenzylpiperidine derivatives were synthesized with two large structures: one residue was attached to the phenyl ring, and two or more residues were attached to the phenyl ring (Figure 4).Seventeen of the twenty-four compounds showed higher MAO-B inhibition than MAO-A except seven compounds (Table 2).Compound S5 showed potent MAO-B inhibition with an IC 50 value of 0.203 µM, followed by S16 (IC 50 = 0.979 µM).In contrast, compound S15 showed the highest MAO-A inhibition with an IC 50 value of 3.691 µM, followed by S5 (IC 50 = 3.857 µM) (Table 2).The selectivity index (SI) of MAO-B over MAO-A was the highest for compound S5 (19.04) and the lowest for compound S9 at 0.62 (Table 2).

Reversibility Studies
In the reversibility study, the concentrations of S5 and S16 used were approximately 2.0 times their IC 50 values (0.4 and 2.0 µM for MAO-B, respectively).The residual activities before and after recovery are represented by A U and A D .Compounds S5 and S16 were recovered from 27.53% to 79.07% and from 29.04% to 81.33%, respectively (Figure 5).The recovery values of S5 and S16 were similar to safinamide (ranging from 28.34% to 81.19%), whereas they differed from pargyline (ranging from 32.66% to 32.93%).These results indicate that S5 and S16 are reversible MAO-B inhibitors.

Enzyme and Inhibition Kinetics
The enzyme kinetics of MAO-B and the types of inhibition were analyzed at five concentrations of benzylamine as a substrate and three inhibitor concentrations.In the LB plots, S5 and S16 appeared to be competitive MAO-B inhibitors (Figure 6A,C).Secondary plots showed that the Ki value was 0.155 ± 0.050 and 0.721 ± 0.074 µM, respectively (Figure 6B

Enzyme and Inhibition Kinetics
The enzyme kinetics of MAO-B and the types of inhibition were analyzed at five concentrations of benzylamine as a substrate and three inhibitor concentrations.In the LB plots, S5 and S16 appeared to be competitive MAO-B inhibitors (Figure 6A,C).Secondary plots showed that the K i value was 0.155 ± 0.050 and 0.721 ± 0.074 µM, respectively (Figure 6B

Enzyme and Inhibition Kinetics
The enzyme kinetics of MAO-B and the types of inhibition were analyzed at five concentrations of benzylamine as a substrate and three inhibitor concentrations.In the LB plots, S5 and S16 appeared to be competitive MAO-B inhibitors (Figure 6A,C).Secondary plots showed that the Ki value was 0.155 ± 0.050 and 0.721 ± 0.074 µM, respectively (Figure 6B

Parallel Artificial Membrane Permeability Assay (PAMPA) for Blood-Brain Barrier (BBB) Permeation Study
The results obtained from the PAMPA indicated significant levels of permeation and CNS bioavailability for compounds S5 and S16.S5 showed a Pe value surpassing 4.0 × 10 −6 cm•s −1 , while S16 came close to this threshold, suggesting higher rates of BBB penetration at 5.75 × 10 −6 cm•s −1 and 3.78 × 10 −6 cm•s −1 , respectively (Table 3).The effective delivery of CNS medications relies on successful brain penetration.To evaluate the brain permeability of all the derivatives, PAMPA-BBB was used in this study.The extent of penetration was determined using a specific formula and considering the effective permeability of the substance (Log Pe).Compounds classified as potentially permeating (CNS+) exhibited a Pe value greater than 4.0 × 10 −6 cm•s −1 , while those categorized as likely non-BBB permeating (CNS−) had a Pe value less than 2.0 × 10 −6 cm•s −1 .

Molecular Docking
Molecular docking allows for the prediction of the molecular affinity at the binding site, important interactions, and experimental binding mechanisms.With this knowledge of the molecular interactions between the physical and chemical processes, new derivatives can be developed.Therefore, we studied the molecular docking of S5 and 2V5Z (Figure 7).The results obtained from the PAMPA indicated significant levels of permeation and CNS bioavailability for compounds S5 and S16.S5 showed a Pe value surpassing 4.0 × 10 −6 cm•s −1 , while S16 came close to this threshold, suggesting higher rates of BBB penetration at 5.75 × 10 −6 cm•s −1 and 3.78 × 10 −6 cm•s −1 , respectively (Table 3).
The effective delivery of CNS medications relies on successful brain penetration.To evaluate the brain permeability of all the derivatives, PAMPA-BBB was used in this study.The extent of penetration was determined using a specific formula and considering the effective permeability of the substance (Log Pe).Compounds classified as potentially permeating (CNS+) exhibited a Pe value greater than 4.0 × 10 −6 cm•s −1 , while those categorized as likely non-BBB permeating (CNS−) had a Pe value less than 2.0 × 10 −6 cm•s −1 .
Table 3. Blood-brain barrier assay of key compounds using PAMPA method.

Molecular Docking
Molecular docking allows for the prediction of the molecular affinity at the binding site, important interactions, and experimental binding mechanisms.With this knowledge of the molecular interactions between the physical and chemical processes, new derivatives can be developed.Therefore, we studied the molecular docking of S5 and 2V5Z (Figure 7).

Chemistry
The initial step in the synthesis was a benzylpiperidine substitution at position six.The pathway begins with the commercially acquired 3,6-dichloropyridazine and proceeds in five steps to obtain the final products.Mass spectral data demonstrated that the substitution was performed unilaterally and that the first-stage product was obtained with a yield of 52%.The lactam carbonyl present in the pyridazinone ring at 1660 cm −1 in the IR

Chemistry
The initial step in the synthesis was a benzylpiperidine substitution at position six.The pathway begins with the commercially acquired 3,6-dichloropyridazine and proceeds in five steps to obtain the final products.Mass spectral data demonstrated that the substitution was performed unilaterally and that the first-stage product was obtained with a yield of 52%.The lactam carbonyl present in the pyridazinone ring at 1660 cm −1 in the IR spectral data was used to confirm the results of the hydrolysis of the pyridazine ring in an acidic environment, which was the following step.The SN2 reaction attacks the bromine-bonded carbon of ethyl bromoacetate, producing an acetate derivative.This attack was caused by the unshared electrons of the nitrogen atom in the second position of the pyridazinone ring.Given the ability of bromine to retain electrons, the second nitrogen of the pyridazinone ring attacks the electron-poor carbon atom in the basic environment produced by the presence of potassium carbonate.The ester derivative and hydrazine hydrate react to produce the acetohydrazide derivative, which was produced in the following steps.As the alcohol is stripped from the molecule, the hydrazine hydrate nucleophile hits the carbonyl carbon, initially causing an addition reaction, followed by an elimination reaction by the formation of a double bond between carbon and oxygen.
The final stage involved the reaction of acetohydrazide with both substituted and unsubstituted benzaldehyde derivatives to produce the final compounds.Similar to the previous synthesis step, the next step involves an elimination reaction in which water is released from the structure and a carbon-nitrogen double bond is formed.The addition reaction starts with the nucleophilic attack of the free electrons of the hydrazine nitrogen on the carbon of the benzaldehyde carbonyl.In derivatives from benzaldehyde derivatives with electron-withdrawing substituents on the ring, the resulting compounds were produced in yields as high as 97.37%.

Molecular Docking
AutoDock Vina software was used for docking.Vina uses PDBQT molecular structure file types as both input and output.Vina created up to 30 configurations for each docking run, ranking them according to the largest energy difference (kcal/mol) between the optimal and lowest binding modes.The optimal docking scores for S5 and safinamide in affinity were −10.4 and −11.0 kcal/mol, respectively.The S5 and safinamide and S5 bound to the same binding pocket with almost similar amino acid residues.In the case of a native ligand, the major interactions were hydrophobic and bipartite hydrogen bonds between the amide side chain and Gln206 residue.The binding study results indicated that a Pi-sulfur bond holds the pyridazine ring of S5 to Cys172.Pro102 and Leu171 form a typical hydrogen bond with S5.Tyr398 and Tyr326 residues utilize pi-pi stacking to interact with the ligand (Figure 7).As noted above, 5S suppressed MAO-B expression in vitro.Furthermore, we observed that hydrogen bonding and hydrophobic interactions stabilized the 2V5Z and S5 complexes.

Chemistry
Scheme 1 shows the preparation of compounds S1-S24.All chemicals were obtained from commercial suppliers.Chloroform-methanol (90:10) was used as the mobile phase in Merck Kieselgel 60 F254 aluminum plates (Darmstadt, Germany), and the reactions were monitored using thin-layer chromatography.The spots were identified under 254 nm UV light.Melting points (mp) were measured without correction using a Thomas-Hoover capillary melting point device (Fredericksburg, VA, USA).An Avonce 600 Ultrashield TM (Bruker, Rheinstetten, Germany) NMR spectrometer was used to record 1H-and 13C-NMR (300 MHz) spectra.Tetramethylsilane was used as an internal reference, and the compounds were dissolved in dimethyl sulfoxide (DMSO-d6) for NMR spectroscopy.The chemical shifts are represented as δ (ppm) values.The terms "singlet", "doublet", "triplet", "quartet", "multiplet", and "doublet of doublet" were used to identify the splitting patterns.Using positive ion (ESI+) and negative ion (ESI−) electrospray ionization procedures, the HRMS spectra of the synthesized compounds were acquired from their solutions in methanol using the Waters LCT Premier XE UPLC/MS TOFF system (Milford, MA, USA) and MassLynx 4.1 software.
solutions in methanol using the Waters LCT Premier XE UPLC/MS TOFF system (Milford, MA, USA) and MassLynx 4.1 software.

An Inhibition Study of MAO-A and MAO-B by the Compounds
In the primary screening, 24 compounds were evaluated for their inhibition of MAO-A or MAO-B at 10 µM concentration.The half-inhibitory concentrations (IC 50 ) of the compounds were calculated using GraphPad Prism software 5 [35].The compounds with IC 50 values of 40 µM or higher were indicated as >40 µM.The selectivity index (SI) was calculated as the IC 50 of MAO-A/IC 50 of MAO-B.The inhibition of compounds was compared with reference inhibitors such as toloxatone and clorgyline (reversible and irreversible inhibitors, respectively) for MAO-A and safinamide and pargyline (reversible and irreversible inhibitors, respectively) for MAO-B inhibitors [35].For the inhibition kinetics, lead compounds were used at three concentrations, approximately 0.5, 1.5, and 2.0 times the IC 50 value [34], and the inhibition constant (K i ) was determined using the secondary plot of their slopes in the LB plots.

Reversibility Studies
The reversibility of the lead compounds to MAO-B was evaluated using a dialysis tube (D-Tube Dialyzer Maxi, MWCO 6-8 kDa, Sigma-Aldrich, St. Louis, MO, USA).Their reversibilities were analyzed by the values of undialyzed (A U ) and dialyzed (A D ) activities, measuring approximately 2.0 times the IC 50 value after pre-incubation for 30 min [34].The reversibility patterns of the lead compounds were determined by comparing A U and A D with those reported in the literature [34].

Blood-Brain Barrier Permeability Study
In early drug studies, a parallel artificial membrane permeation approach was employed to predict the passive transcellular permeability of a drug through the blood-brain barrier (BBB).This involved setting up a sandwich-like structure in the PAMPA using a microtiter plate with 96 wells and a Millipore filter plate with 96 wells (IPVH, 125 m thick filter, 0.45 m pore), which were then submerged in 0.1 mL of n-dodecane.Drug samples were initially prepared as stock solutions in DMSO at 10 mm doses and stored at 0 • C. Before being introduced into a 96-well filter plate, these stock solutions were further diluted at pH 7.4 to achieve final sample concentrations of 0.01, 0.1, 0.5, and 1 mM, with the DMSO content limited to 1% (v/v).The resulting diluted solutions were then transferred to the donor wells (270 µL each), with 200 µL of pH 7.4 buffering agent added to the acceptor well.To create the "sandwich", the donor plate (with the analyte at the bottom, an aqueous recipient on top, and an artificial lipid barrier in the middle) was accurately positioned over the acceptor filter plate.The drug material diffused from the donor well into the acceptor well through the lipid membrane, with the "sandwich" structure remaining intact throughout the process.UV spectroscopy was used to measure the drug concentration in the donor, recipient, and reference wells, and the extent of permeation was assessed using a specific expression [36].

Molecular Docking
The molecular docking software AutoDock Vina and the PDBQT molecular structure file format were used [37].Consequently, AutoDockTools-1.5.6, from the MGLTools-1.5.6 package, was used to create ligands and receptors in the PDBQT file format.ChemDraw 23 was used to draw the S5 chemical structure in pdf format.Furthermore, Chembio3D pro was utilized for optimization, for improved molecular confirmation, using energy minimization with a 0.01 RMS gradient.Three-dimensional ligands were corrected by substituting hydrogen atoms in the water molecules.The crystal structure of 2V5Z was obtained from the Protein Data Bank.Following the extraction of the co-crystallized ligands, ions, and water molecules, a 40 × 40 × 40 grid box with a grid spacing of 0.375, X = 51.901,Y = 156.468,and Z = 28.561 was formed.The receptor file [38] was saved in the PDBQT file format.Vina was run on a Windows 10 PC with a Core i5 processor.A standard configuration file was established to specify grid box coordinates, exhaustiveness, computer usage, and output data for the docking experiment.The search was set to eight intensities to cover as much of the ground as possible.The docking experiments were initiated using a command line.
The docking experiment produced a PDBQT file with 20 different docked ligand positions on the receptor.The ideal position was selected by considering the docking score, number of H-bonds, and visual inspection of each docking position.The PDB format was used to store the selected receptor and ligand positions.The resulting PDB file was examined for ligand-receptor interactions using LigPlot + version 2.2 [39,40] and Biovia Discovery Studio 2021.

Conclusions
Twenty-four derivatives of pyridazinobenzylpiperidine were prepared, and their ability to inhibit monoamine oxidase (MAO) was assessed.The compound displayed greater MAO-B than MAO-A inhibition.Compound S15 demonstrated the most potent MAO-A inhibition, whereas compounds S5 and S16 demonstrated significant competitive and reversible MAO-B inhibition.Overall, if we look into the selectivity index, we can conclude that most of the compounds showed non-specific MAO inhibition.Additionally, BBB penetration was demonstrated by compounds S5 and S16, and molecular docking experiments indicated that compound S5 stabilized the protein-ligand complex.Based on these findings, lead compounds may be used as medications to treat neurological conditions.

Figure 1 .
Figure 1.The metabolism of dopamine.

Figure 2 .
Figure 2. The molecular structures of selegiline, rasagiline, and safinamide, which are MAO-B inhibitors currently used in the treatment of PD.

Figure 1 .
Figure 1.The metabolism of dopamine.

Figure 1 .
Figure 1.The metabolism of dopamine.

Figure 2 .
Figure 2. The molecular structures of selegiline, rasagiline, and safinamide, which are MAO-B inhibitors currently used in the treatment of PD.

Figure 2 .
Figure 2. The molecular structures of selegiline, rasagiline, and safinamide, which are MAO-B inhibitors currently used in the treatment of PD.

Figure 4 .
Figure 4.The design targets to be applied on the molecule within the scope of this study.The pyridazinone ring is protected as the main structure (red), containing the benzyl moiety at the 6th position of pyridazinone (blue), and hydrazone part (orange) and substituted phenyl moiety (green).

Figure 4 .
Figure 4.The design targets to be applied on the molecule within the scope of this study.The pyridazinone ring is protected as the main structure (red), containing the benzyl moiety at the 6th position of pyridazinone (blue), and hydrazone part (orange) and substituted phenyl moiety (green).

Figure 4 .
Figure 4.The design targets to be applied on the molecule within the scope of this study.The pyridazinone ring is protected as the main structure (red), containing the benzyl moiety at the 6th position of pyridazinone (blue), and hydrazone part (orange) and substituted phenyl moiety (green).

Molecules 2024, 29 , 3097 6 of 19 Figure 5 .
Figure 5.The recovery of MAO-B inhibition by S5 and S16 using dialysis experiments.The concentrations of S5 and S16 used were approximately 2.0 times their IC50 values (0.4 and 2.0 µM).After 30 min of pre-incubation, the mixtures were dialyzed for 6 h with two buffer changes.

Figure 6 .
Figure 6.Lineweaver-Burk (LB) plots for MAO-B inhibition by S5 and S16 (A,C), and their secondary plots (B,D) of the slopes versus inhibitor concentrations.The experiments were analyzed using five substrate concentrations and three inhibitor concentrations.

Figure 5 .
Figure 5.The recovery of MAO-B inhibition by S5 and S16 using dialysis experiments.The concentrations of S5 and S16 used were approximately 2.0 times their IC 50 values (0.4 and 2.0 µM).After 30 min of pre-incubation, the mixtures were dialyzed for 6 h with two buffer changes.

19 Figure 5 .
Figure 5.The recovery of MAO-B inhibition by S5 and S16 using dialysis experiments.The concentrations of S5 and S16 used were approximately 2.0 times their IC50 values (0.4 and 2.0 µM).After 30 min of pre-incubation, the mixtures were dialyzed for 6 h with two buffer changes.

Figure 6 .
Figure 6.Lineweaver-Burk (LB) plots for MAO-B inhibition by S5 and S16 (A,C), and their secondary plots (B,D) of the slopes versus inhibitor concentrations.The experiments were analyzed using five substrate concentrations and three inhibitor concentrations.

Figure 6 .
Figure 6.Lineweaver-Burk (LB) plots for MAO-B inhibition by S5 and S16 (A,C), and their secondary plots (B,D) of the slopes versus inhibitor concentrations.The experiments were analyzed using five substrate concentrations and three inhibitor concentrations.

Table 1 .
Structures, yields, and chemical-physical data of titled compounds.

Table 1 .
Structures, yields, and chemical-physical data of titled compounds.

Table 2 .
The inhibition of MAO-A and MAO-B by the S series a .
a Results are means ± standard errors of duplicate or triplicate experiments.b Selectivity index (SI) values are expressed as IC 50 of MAO-B versus IC 50 of MAO-A.

Table 3 .
Blood-brain barrier assay of key compounds using PAMPA method.